Characterization of human beta-interferon-binding sites on human cells.

M R Thompson,Z Zhang,A Fournier,Y H Tan

doi:10.1016/s0021-9258(18)89769-1

Abstract

Radioiodinated recombinant human beta-interferon (rHuIFN beta Ser), with almost full (greater than 90%) biological activity, was used to study the binding of human beta-interferon to Daudi cells. Specific binding was not observed with less biologically active (less than or equal to 10%) radioiodinated interferon. The bound radioiodinated interferon was shown to compete with human beta-interferon (HuIFN beta), rHuIFN beta Ser, human alpha-interferon (HuIFN alpha) and with human gamma-interferon (HuIFN gamma). Scatchard plot analyses suggest the presence of about 10,000 binding sites for HuIFN beta/Daudi cell. About 6,600 of these sites can be blocked by HuIFN alpha and 3,700 sites can be blocked by HuIFN gamma. The apparent Kd for HuIFN beta is 2.7 nM. The apparent Kd values for HuIFN alpha and HuIFN gamma are 3.7 and 1.1 nM, respectively. It was possible to demonstrate the cross-linking of HuIFN beta to two macromolecular components of Mr = 128,000 and 103,000. We propose the existence of at least two binding sites for HuIFN beta in Daudi cells, one site recognizing both HuIFN beta and HuIFN gamma, the other site recognizing both HuIFN beta and HuIFN alpha. Each site is capable of recognizing only HuIFN gamma or HuIFN alpha.

Highlights

From the Dewartments of Microbiolom and Medical Biochemistry, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canhda T2N 4 N l
The bound radioiodinated interferon was shown to compete with human &interferon (HuIFNB), rHuIFN&,human a-interferon (HuIFNa) and with human y-interferon (HuIFNy).Scatchard plot analyses suggest the presence of about 10,000 binding sites for HuIFN@/Daudicell
About 6,600 of these sites can be blocked by HuIFNa and 3,700 sites can be blocked by HuIFNr

Summary

RESULTS

Hiological activity.T h e radiospecific activity varied from150- Sepharose column. which separates Inheled I F S from Ihc. low-molrc-. Which separates Inheled I F S from Ihc. low-molrc-. 450 cpm/unit, compared with - 0 cpm/unit ohtained hy the rrlar-weight reartants used durinx radioic~dinntic~rln; nr,s rl and v are similar t o lnnc c, except that the Inheled I F S nre from fractions rlutc*d. -70 "C both '""I-laheled HulFNIaj nd the hand corresponding t o the monomeric f. V , = 17.000) and tctrzlrHuIFN&,., retained at least 90% of their biological activity meric ( M , = fiH.000) forms o f laheled I F S. The radioactivity bound to these items is counted in a LKB Rackgamma counter after the cells and plasticare washed as described under"Experimental Procedures." The figures listed below are mean f S.E. of three samples. Radioactivity bound in Absence of Presence of 100-fold unlabeled HuIFNB unlabeled HuIFNS " cpm

Daudi cells Daudi cells*

Findings

CompetitionbetweenVariousInterferonsforBinding to